Many important zoonotic arboviruses utilize mosquito vectors in the Culex (Melanoconion) subgenus, and one particular group (the Spissipes Section, 23 species) includes most of these vectors in the neotropics. A major impediment to epidemiological studies, including those of the parent grant, is the difficulty in identifying and classifying mosquitoes in this difficult group. We also have preliminary evidence from DNA sequences for a new, cryptic Melanoconion vector species. These results raise important questions: What are the evolutionary relationships within the Spissipes Section and between the Spissipes and other Melanoconion mosquito sections? Has the Spissipes Section co-evolved (cospeciated) with any arboviruses? Do additional cryptic species remain undetected, including important vectors? These issues will be addressed in 2 specific aims: 1. Examine the evolutionary relationships within the Culex (Melanoconion) subgenus and among subgenera of Culex, with emphasis on vectors in the Spissipes Section, using ribosomal and mitochondrial DNA sequencing and phyloglenetic methods. 2. Develop molecular diagnostic tools, based on PCR amplification of ITS-2 ribosomal DNA sequences for rapid identification of important vector species within the Culex (Melanoconion) subgenus. This project will strengthen and extend collaborative interactions between Dr. Scott Weaver, an arbovirologist and vector biologist from the University of Texas Medical Branch, and Dr. Juan Carlos Navarro, a mosquito systematist from the Institute of Tropical Zoology Caracas, Venezuela. These studies will enhance many epidemiological studies of Neotropical arboviruses and will elucidate the evolution of arbovirus- vector interactions. Our data will form the cornerstone for future work on the genetic, ecological and/or physiological factors that regulate arbovirus transmission by specific mosquito groups such as the Spissipes section of the subgenus Melanoconion.